//Works with ImageJ 1.52n and Fiji
//Bio-formats release version: 5.2.1
//Developed using Nikon nd2 files
//Only works for 2 or 3 channel pictures currently
//Pictures don't need to be split
//Make sure there are no other images open.
//You might as well make the Bio-formats "windowless" since it will pop up every time that you open a picture.

//This is version 2.0 of this macro. Updated 04/22/2019

waitForUser("Make sure that you do not have any images open that need to be saved.\nThe macro will close them all if not.");

//This allows this macro to be utilized for other measurements.
//There is a "setOption" parameter down in the ROI section that forces the "display label" so that all measurements are labeled with the picture name.
run("Set Measurements...");

//this is the directory with your pictures.
//make sure there are no subfolders!
dir1 = getDirectory("Choose your folder with your original pictures");
list = getFileList(dir1);  //for the batch to work
setBatchMode(true);
run("Close All");
open(dir1+list[0]);
if(nImages==1){
Imagessplit=0; //0 = no

getDimensions(w, h, channels, slices, frames);
  Stack.getPosition(channel, slice, frame);
  //print("   Channels: "+channels);

}
else if(nImages>1){channels=nImages;
//print(channels);
Imagessplit=1; //1 = yes

}
run("Close All");
setBatchMode(false);

Total = channels;
if(channels>3){
	waitForUser("This macro does not work with images that have less than two or more than three.\nThis image has "+channels+" channels.");
}
if(channels<2){
	waitForUser("This macro does not work with images that have less than two or more than three.\nThis image has "+channels+" channels.");
}
//dialog boxes -which channels need threshold
Dialog.create("Threshold");

  Dialog.addMessage("Which channels need to a threshold?");

 Dialog.addCheckbox("Channel 1", true);
 if(Total>=2) {
	Dialog.addCheckbox("Channel 2", true);
}

if(Total==3) {
 Dialog.addCheckbox("Channel 3", false);
}

 Dialog.show();
  Ch1 = Dialog.getCheckbox();
  Ch2 = Dialog.getCheckbox();;
if(Total==3) 
{Ch3 = Dialog.getCheckbox();;;}
 
else Ch3=0;

if(Total==3) totalchan=3;
 else if(Total>=2) totalchan=2; 
 else totalchan=1;

//dialog boxes -what are the threshold values
Dialog.create("Threshold Values");
if(Ch1==true) {
	Dialog.addNumber("Channel 1 Lower Threshold value:", 900);
}
if(Ch2==true) {
	Dialog.addNumber("Channel 2 Lower Threshold value:", 900);
}
if(Ch3==true) {
	Dialog.addNumber("Channel 3 Lower Threshold value:", 1000);
}
  Dialog.show();

if(Ch1==true) {
	Lowch1=Dialog.getNumber();
} 

if(Ch2==true) {
	Lowch2=Dialog.getNumber();;
}
if(Ch3==true) {
	Lowch3=Dialog.getNumber();;;
}
Dialog.create("Analysis");

 Dialog.addMessage("Which channels need to be measured?");

 Dialog.addCheckbox("Ch. 1", true);
 if(Total>=2) {
Dialog.addCheckbox("Ch. 2", true);
}

if(Total==3) {
Dialog.addCheckbox("Ch. 3", false);
}

Dialog.show();
Ch1measure = Dialog.getCheckbox();
Ch2measure = Dialog.getCheckbox();;
if(Total==3) 
{Ch3measure = Dialog.getCheckbox();;;}
 
else Ch3measure=0;

//batch opening stuff goes here:

list = getFileList(dir1);  //for the batch to work
for (i=0; i<list.length; i++) { //for the batch to work
open(dir1+list[i]); //for the batch to work
	
//checks whether channels are split or not and splits them
if(Imagessplit==0)
{
	originaltitle=getTitle();
	
	run("Split Channels");
	totalchannels=nImages();
	
	if(totalchan!=totalchannels){
		print("Channel numbers are not the same for all the pictures");
		waitForUser("Please make sure all the images have the same number of channels.\nThe macro is currently set for "+totalchan+" channels.\nThis image has "+totalchannels+" channels.");
		kill //this isn't a real function. it will just kill the macro or at least through it to the debug screen.
	
	}
	if(totalchannels>=2)
	{
		selectWindow("C2-"+originaltitle);
		rename(originaltitle+" - C=1");
		C1=getTitle();
	}
	if(totalchannels==3)
	{
		selectWindow("C3-"+originaltitle);
		rename(originaltitle+" - C=2");
		C2=getTitle();
	}
	selectWindow("C1-"+originaltitle);
	rename(originaltitle+" - C=0");
	C0=getTitle();
}
if(Imagessplit==1)
{
	if(totalchan!=totalchannels){
		print("Channel numbers are not the same for all the pictures");
		waitForUser("Please make sure all the images have the same number of channels.\nThe macro is currently set for "+totalchan+" channels.\nThis image has "+totalchannels+" channels.");
		kill //this isn't a real function. it will just kill the macro or at least through it to the debug screen.
	
	}
	originaltitle=File.name;
	totalchannels=nImages();
	if(totalchannels>=2)
	{
		selectWindow(originaltitle+" - C=1");
		C1=getTitle();
	}
	if(totalchannels==3)
	{
		selectWindow(originaltitle+" - C=2");
		C2=getTitle();
	}
	selectWindow(originaltitle+" - C=0");
	rename(originaltitle+" - C=0");
	C0=getTitle();
}
//start of individual picture analysis
selectWindow(C0);
run("Z Project...", "projection=[Max Intensity]");
C0max=getTitle();
selectWindow(C0);
close();

if(totalchannels>=2)
{
	selectWindow(C1);
	run("Z Project...", "projection=[Max Intensity]");
	C1max=getTitle();
selectWindow(C1);
	close();
}
if(totalchannels==3)
{
	selectWindow(C2);
	run("Z Project...", "projection=[Max Intensity]");
	C2max=getTitle();
	selectWindow(C2);
	close();
}

//threshold
if(Ch3==true)
{
	selectWindow(C2max);
	setAutoThreshold("Default dark");
// run("Threshold...");

getThreshold(lower, upper);
setThreshold(Lowch3, upper);

setOption("BlackBackground", true);
run("Convert to Mask");
}

if(Ch2==true)
{
	selectWindow(C1max);
	setAutoThreshold("Default dark");
// run("Threshold...");

getThreshold(lower, upper);
setThreshold(Lowch2, upper);


setOption("BlackBackground", true);
run("Convert to Mask");
}

if(Ch1==true)
{
	selectWindow(C0max);
		setAutoThreshold("Default dark");
// run("Threshold...");

getThreshold(lower, upper);
setThreshold(Lowch1, upper);


setOption("BlackBackground", true);
run("Convert to Mask");
}
waitForUser("Please find your ROI.\nClick \"OK\" once you have.");
n = roiManager("count");
if(n == 0) {
	waitForUser("No ROI found.\nPlease find your ROI.\nClick \"OK\" once you have.");
}

setOption("Display label", true); //this is here to make sure that each measurement is labeled with the picture it came from. Comment it out if you don't want it

if(Ch1measure==true){
selectWindow(C0max);	
//run("Set Measurements...", "area integrated display redirect=None decimal=3");
rename(C0);
  	
	roiManager("Measure");

	}


if(Ch2measure==true){
	
selectWindow(C1max);	
//run("Set Measurements...", "area integrated display redirect=None decimal=3");
rename(C1);	
  	
	roiManager("Measure");

	}

if(Ch3measure==true){
	
selectWindow(C2max);
rename(C2);	
//run("Set Measurements...", "area integrated display redirect=None decimal=3");

  	
	roiManager("Measure");

}
run("Close All");
roiManager("Deselect");
roiManager("Delete");

	}//needed for batch